Species-Specific Discrimination of Insect Meals for Aquafeeds by Direct Comparison of Tandem Mass Spectra

Insect protein has the potential to become a sustainable feed ingredient for the rapidly growing aquaculture industry. In the European Union, insect derived protein is placed under the same legislation as processed animal proteins (PAP). It is therefore of interest to develop methods for regulatory use, which unambiguously identify the species origin of insect-based ingredients. We performed (i) total protein quantification of insect samples using the traditional nitrogen-to-protein conversion factor of 6.25 and the sum of anhydrous amino acids, (ii) quantitative amino acid profiling and (iii) high-throughput tandem mass spectrometry to describe and differentiate 18 different commercial-grade insect meal samples derived from Hermetia illucens (8), Tenebrio molitor (5), Alphitobius diaperinus (3) and Acheta domesticus (2). In addition, we investigated and compared different protein extraction and digestion protocols for proteomic analysis. We found that irrespective of sample preparation, shotgun proteomics in combination with direct spectral comparison were able to differentiate insect meal according to their taxonomic classification. The insect specific spectral libraries created in the present work can in future be used to develop more sensitive targeted methods of insect PAP identification and quantification in commercial feed mixtures

Future feed control – Tracing banned bovine material in insect meal

In the present study, we assessed if different legacy and novel molecular analyses approaches can detect and trace prohibited bovine material in insects reared to produce processed animal protein (PAP). Newly hatched black soldier fly (BSF) larvae were fed one of the four diets for seven days; a control feeding medium (Ctl), control feed spiked with bovine hemoglobin powder (BvHb) at 1% (wet weight, w/w) (BvHb 1%, w/w), 5% (BvHb 5%, w/w) and 10% (BvHb 10%, w/w). Another dietary group of BSF larvae, namely *BvHb 10%, was first grown on BvHb 10% (w/w), and after seven days separated from the residual material and placed in another container with control diet for seven additional days. Presence of ruminant material in insect feed and in BSF larvae was assessed in five different laboratories using (i) real time-PCR analysis, (ii) multi-target ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS), (iii) protein-centric immunoaffinity-LC-MS/MS, (iv) peptide-centric immunoaffinity-LC-MS/MS, (v) tandem mass spectral library matching (SLM), and (vi) compound specific amino acid analysis (CSIA). All methods investigated detected ruminant DNA or BvHb in specific insect feed media and in BSF larvae, respectively. However, each method assessed, displayed distinct shortcomings, which precluded detection of prohibited material versus non-prohibited ruminant material in some instances. Taken together, these findings indicate that detection of prohibited material in the insect-PAP feed chain requires a tiered combined use of complementary molecular analysis approaches. We therefore advocate the use of a combined multi-tier molecular analysis suite for the detection, differentiation and tracing of prohibited material in insect-PAP based feed chains and endorse ongoing efforts to extend the currently available battery of PAP detection approaches with MS based techniques and possibly δ13CAA fingerprinting.publishedVersio

ALADIN laser frequency stability and its impact on the Aeolus wind error

The acquisition of atmospheric wind profiles on a global scale was realized by the launch of the Aeolus satellite, carrying the unique Atmospheric LAser Doppler INstrument (ALADIN), the first Doppler wind lidar in space. One major component of ALADIN is its high-power, ultraviolet (UV) laser transmitter, which is based on an injection-seeded, frequency-tripled Nd:YAG laser and fulfills a set of demanding requirements in terms of pulse energy, pulse length, repetition rate, and spatial and spectral beam properties. In particular, the frequency stability of the laser emission is an essential parameter which determines the performance of the lidar instrument as the Doppler frequency shifts to be detected are on the order of 10^8 smaller than the frequency of the emitted UV light. This article reports the assessment of the ALADIN laser frequency stability and its influence on the quality of the Aeolus wind data. Excellent frequency stability with pulse-to-pulse variations of about 10 MHz (root mean square) is evident for over more than 2 years of operations in space despite the permanent occurrence of short periods with significantly enhanced frequency noise (> 30 MHz). The latter were found to coincide with specific rotation speeds of the satellite's reaction wheels, suggesting that the root cause are micro-vibrations that deteriorate the laser stability on timescales of a few tens of seconds. Analysis of the Aeolus wind error with respect to European Centre for Medium-Range Weather Forecasts (ECMWF) model winds shows that the temporally degraded frequency stability of the ALADIN laser transmitter has only a minor influence on the wind data quality on a global scale, which is primarily due to the small percentage of wind measurements for which the frequency fluctuations are considerably enhanced. Hence, although the Mie wind bias is increased by 0.3 m/s at times when the frequency stability is worse than 20 MHz, the small contribution of 4 % from all Mie wind results renders this effect insignificant (< 0.1 m/s) when all winds are considered. The impact on the Rayleigh wind bias is negligible even at high frequency noise. Similar results are demonstrated for the apparent speed of the ground returns that are measured with the Mie and Rayleigh channel of the ALADIN receiver. Here, the application of a frequency stability threshold that filters out wind observations with variations larger than 20 or 10 MHz improves the accuracy of the Mie and Rayleigh ground velocities by only 0.05 and 0.10 m/s, respectively, however at the expense of useful ground data

Determination of the ruminant origin of bone particles using fluorescence in situ hybridization (FISH).

Molecular biology techniques such as PCR constitute powerful tools for the determination of the taxonomic origin of bones. DNA degradation and contamination by exogenous DNA, however, jeopardise bone identification. Despite the vast array of techniques used to decontaminate bone fragments, the isolation and determination of bone DNA content are still problematic. Within the framework of the eradication of transmissible spongiform encephalopathies (including BSE, commonly known as "mad cow disease"), a fluorescence in situ hybridization (FISH) protocol was developed. Results from the described study showed that this method can be applied directly to bones without a demineralisation step and that it allows the identification of bovine and ruminant bones even after severe processing. The results also showed that the method is independent of exogenous contamination and that it is therefore entirely appropriate for this application